Name: electrophysiological methods for measuring photopigment levels in drosophila photoreceptors
Uploaded: 2022-06-02T12:40:00
Description: Watch this Scientific Journal Video about Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors at JoVE.com

这项研究得到了以色列科学基金会（ISF）和美国 - 以色列双国科学基金会（BSF）的资助。我们感谢阿纳托利·沙波奇尼科夫先生建造蜡丝加热器。

1. 使用视网膜电图测量强度反应关系、延长去极化后电位 （PDA） 和早期受体电位 （ERP）
<ol><li>适合 黑腹角鲷 制备的养殖条件<ol>
<li>饲养 的黑腹产儿 在含有标准黄玉米的瓶子中飞行，该瓶子含有食物，在温度保持在24°C和12小时的暗/光循环中</li>
		<li>在实验前至少24小时将苍蝇瓶保持在黑暗中。</li>
	</ol></li>
	<li>常规设置<ol>
<li>通过拉动1 mm x 0.58 mm（外径x I.D）纤维填?...

<ol>
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使用 果蝇 光感受器制剂的主要优点是其可访问性，光刺激的易用性和准确性，最重要的是，应用分子遗传学能力的能力7。广泛的遗传学研究已经确定 果蝇 是复杂生物过程遗传解剖的非常有用的模型系统7。 果蝇 基因组结构相对简单（仅由四条染色体组成，包括X和Y性染色体，两个较大的常染色体元素，2号和3号染色体以及小点第四条染色?...

光激活的视紫红质（R）是一种G蛋白偶联受体（GPCR），由7个跨膜蛋白（视蛋白）和一个发色团组成。在 果蝇 （果蝇）中， 光子吸收诱导11-顺式-3-OH-视网膜发色团异构化为全反式-3-OH-视网膜1，促进视紫红质到偏视紫红质的构象变化（M， 图1A）。与脊椎动物视紫红质不同，无脊椎动物发色团的主要部分不会与视蛋白解离，从而产生生理活性的暗稳定色素状态M。反过来，全反式-3-OH-视网膜发色团的额外光子吸收诱导发色团2，3的异构化，产生具有11-顺式-3-OH-视网膜发色团的R色素状态。R状态是一种深色，稳定且生理上无活性的光色素。除了发色团4的极快光子再生途径（与脊椎动物光色素沉着非常相似）之外，无脊椎动物中还存在用于发色团再生的替代酶促慢速途径，其中一些阶段在光感受器细胞周围的视网膜细胞中进行5，6。
果蝇作为研究无脊椎动物光感受器的模式生物具有很大的优势。特别是，制剂的可及性和应用分子遗传学的能力使果蝇成为一个强大的模型系统7。因此，已经建立了几种体内和离体实验方法，用于研究光转导的一般情况，特别是光色素水平。最简单的体内方法利用了相对较小的细胞外记录的对果蝇眼光的电压响应。因此，光刺激唤起整个眼睛的电压响应，可以使用细胞外视网膜电图（ERG）记录来测量，这比脊椎动物眼睛的光的ERG响应8，9大约3个数量级。果蝇ERG反应是稳健的，易于获得，这使其成为识别由于突变引起的光反应异常的便捷方法。对光的ERG反应主要来自光感受器，色素（神经胶质细胞）细胞和层的次级神经元（见图1B）。ERG的主要成分是（i）光感受器的细胞外电压响应，（ii）从层神经元产生的光刺激开始和结束时的“开”和“关”瞬变（图2A，插入，ON，OFF），（iii）神经胶质细胞的缓慢响应（图2A，插图，箭头），以及（iv）短暂和瞬态响应， 由ON瞬变10之前的光色素激活期间的电荷位移引起（图2C [插图]，D，E）。这种短暂的反应由两相（M1和M2，图2C[插图]）组成，只能通过极强的光刺激诱导，同时激活数百万个光色素分子。在蓝色刺激下（图2D，蓝色迹线）和具有高度降低的光色素水平的突变体（图2E，红色迹线）中既未观察到，但在消除PLC活性的突变体中，其振幅轻度增强（图2E，橙色迹线）。M1相是苍蝇的典型ERP，由光感受器中M的激活引起。具有正极性（细胞内）的M1相以正常方式在符号反转突触中释放神经递质并激活层神经元，这些神经元通过产生突触放大的M2相来响应光感受器去极化。因此，M1和M2相位都反映了M活化10，11。
光感受器的去极化产生角膜阳性“开启”瞬时，由光感受器轴突和层10，11的单极神经元之间的符号反转突触引起（图1B）。ERG的缓慢上升和衰减是由色素细胞的去极化引起的（图2A，插图，箭头），主要是由于K +通过瞬时受体电位（TRP）和TRP样（TRPL）通道13，14，15从感光细胞12流出。与感光器对光的细胞内或全细胞记录相比，这些缓慢的动力学成分在很大程度上掩盖并扭曲了光感受器反应的波形9，10。此外，在非常强的照明下，可以观察到额外的瞬态响应，该响应在“导通”瞬变之前并部分融合（图2C [插图]，D，E）。该信号直接起源于光色素10的大量激活。
已经开发了几种使用中性密度（ND）和彩色滤光片的光照方案，以及强烈的照明闪光灯，以研究眼睛的一般情况，特别是光转导级联。这些方案也被用于研究光色素的性质。
强度响应协议测量整个眼睛对增加的光强度的ERG电压响应的峰值幅度（图2A，B）。该协议有助于检测光感受器细胞对光9的敏感性的变化。
延长去极化后电位（PDA）方案利用了视紫红质和偏视紫红质吸收光谱的差异，允许在果蝇中将R的大量光色素转化为其生理活性和暗稳定的中间M状态2。在ERG电压响应中，给出相对较短的饱和光脉冲，并记录产生的电压响应。在这种情况下，去极化信号达到上限（反转电位），因为激活大量视紫红质分子（~1×108）的一小部分足以达到天花板。光转导分量的大量存在确保了即使在浓度显着降低或光传导分量的细微故障的突变体中也能达到该上限。这种情况排除了这些突变体的隔离。Pak等人介绍了PDA筛查7，寻求一种可靠且揭示性的测试来分离视觉突变体。在果蝇中，PDA反应是通过遗传去除红色筛选色素（允许光色素转化）和蓝光的应用引起的，蓝光优先被视紫红质吸收（图3A），因此导致R到M光色素状态的大量净转化。光转导终止在光色素水平上被R到M的大净转换破坏，这反过来又导致光关闭后很长一段时间内的持续激发（图2C，图4A [顶部]）。在PDA期间，光感受器对随后的测试灯不太敏感，并且部分脱敏（失活）。PDA甚至可以检测视紫红质生物发生的微小缺陷，并测试光感受器细胞在长时间内保持兴奋的最大能力。由于它严格依赖于高浓度视紫红质的存在，因此很容易对光转导组分的缺乏补充进行评分。值得注意的是，PDA屏幕已经产生了许多新的和非常重要的视觉突变体（在Pak等人中回顾）。因此，由Pak等人分离的PDA突变体对于分析果蝇视觉系统仍然非常有用。
PDA通过饱和蓝光在 果蝇 中诱导，导致光偏移后很长一段时间内的连续去极化（图4A [顶部]）。在饱和PDA诱导蓝光后，外周光感受器（R1-6）在其最大容量下在黑暗中保持连续活性，达到饱和。PDA期间额外的饱和蓝光不会在R1-6细胞中产生任何额外的反应，而是在叠加在PDA上的R7-8电池中诱导反应。叠加反应可以通过这些细胞中表达的光色素的不同吸收光谱（R7-8）16来解释。PDA可以通过在饱和橙色光下将M光转换回R来抑制（图4A [顶部]）。PDA将感光细胞带到其最大活性能力的能力，这种情况无法通过强烈的白光实现，这解释了为什么它一直是筛选 果蝇视觉突变体的主要工具。这是因为它允许检测参与正常光色素水平17，18的生物发生的蛋白质中的微小缺陷。已经分离出两组PDA缺陷突变体：既不失活也不后电位（nina）突变体和失活但不是后电位（ina） 突变体。前者的表型是缺乏PDA和由光色素水平大幅降低引起的相关失活（图4A [中]）。后者的表型显示灭活，但蓝光后没有暗去极化，因为突变体中具有正常的视紫红质水平，但缺乏与TRP通道相互作用的蛋白质（图4A [底部]）。
PDA由相对于逮捕素（ARR2）的光色素量的差异引起，其结合并终止M活性19，20，21（图1A）。在果蝇光感受器中，光色素的量大约是ARR219量的五倍。因此，ARR2水平不足以灭活由R到M的大净光转换产生的所有M分子，留下过量的M在黑暗中不断活跃17，19，20，22，23。该机制解释了通过突变或类胡萝卜素剥夺24，25消除PDA反应，导致光色素水平降低，但不影响逮捕素水平。此外，这种解释还解释了空ARR2（arr23）突变等位基因21的表型，其中PDA可以在~10倍变暗的蓝光强度下实现19，20，21（图4B，C）。PDA不是苍蝇光感受器的独特特征，它出现在每个具有暗稳定M的测试物种中，其吸收光谱与R状态的吸收光谱不同，允许光色素从R到M状态的充分光转换。发现PDA现象学的彻底研究物种是藤壶（Balanus）光感受器，其中R状态的吸收光谱比M状态2的波长更长（图3B）。因此，与苍蝇中的情况不同，在藤壶中，橙红色光诱导PDA，而蓝光抑制PDA2。
早期受体电位（ERP）方案利用了R或M激活期间发生的电荷位移。视觉色素是脊椎动物和无脊椎动物膜3的信号室表面膜的组成部分。因此，在活化过程中，光色素分子从一种中间状态变为下一种中间状态伴随着电荷位移4，26。由于光色素分子与膜电容4平行电排列，快速同步的构象变化产生表面膜的快速极化变化，这在苍蝇中发生在由约30，000-50，000微绒组成的信号室中，称为rhabdomere。然后，这种极化通过细胞体的膜电容被动地放电，直到细胞膜同样极化。ERP是电荷位移的细胞外记录。细胞内记录的ERP表现为由细胞膜的时间常数4，27，28积分的细胞外ERP。由视觉色素电荷位移激活的电流也可以在全电池电压钳记录29，30（图5A-D）中测量，其主要优点（在早期受体电流（ERC）记录中）是最小化膜电容对信号动力学的影响。
协议部分描述了如何通过果蝇分离的卵黄体31，32的全细胞记录对果蝇眼9进行ERG测量和ERC测量。我们还描述了用于研究光转导的特定协议，特别是光色素。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe with elongated tip</td>
			<td></td>
			<td></td>
			<td>Figure 6M</td>
		</tr>
		<tr>
			<td>1 rough tweezers</td>
			<td></td>
			<td>Dumont #5, Standard</td>
			<td>0.1 mm x 0.06 mm, length 110 mm, Inox (Figure 6H)</td>
		</tr>
		<tr>
			<td>2 condenser lenses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A/D converter</td>
			<td>Molecular Device</td>
			<td>Digidata 1200</td>
			<td>Possible replacement: any digidata from molecular devices (e.g 1440A) -Figure 7C</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Almost perfect electronics</td>
			<td></td>
			<td>Possible replacement: Warner instruments- IE251A or IE-210 (comes with headstage)- Figure 7D</td>
		</tr>
		<tr>
			<td>Anti-vibration Table</td>
			<td>Newport</td>
			<td>VW-3036-OPT-01</td>
			<td>Figure 7H</td>
		</tr>
		<tr>
			<td>Capillaries</td>
			<td>Harvard Apparatus</td>
			<td>Borosilicate glass capillaries</td>
			<td>1 mm x 0.58 mm (Figure 6O)</td>
		</tr>
		<tr>
			<td>Clampex</td>
			<td>Molecular Device</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>CO2 tank</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cold light source</td>
			<td>Schott</td>
			<td>KL1500 LCD</td>
			<td>Figure 6C</td>
		</tr>
		<tr>
			<td>Delicate wipers</td>
			<td>Kimtech</td>
			<td></td>
			<td>Kimwipes (Figure 6K)</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td></td>
			<td></td>
			<td>Suitable for capillary O.D. 1 mm (Figure 6N, Figure 7N, and Figure 7P)</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Home made</td>
			<td></td>
			<td>Electromagnetic noise shielding and black front curtain (Figure 7K)</td>
		</tr>
		<tr>
			<td>Filter (Color)</td>
			<td>Schott</td>
			<td>OG590, Edge filter</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Filter (Color)</td>
			<td>Schott</td>
			<td>BP450/40 nm</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Filter (Color)</td>
			<td>Blazers</td>
			<td>550 nm</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Filter (Color) for cold light source</td>
			<td>Schott</td>
			<td>RG630</td>
			<td>Figure 6C</td>
		</tr>
		<tr>
			<td>Filter (Heat)</td>
			<td>Schott</td>
			<td>KG3</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Filters (Neutral density filter)</td>
			<td>Chroma</td>
			<td>6,5,4,3,2,1,0.5,0.3</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Flash Lamp system</td>
			<td>Honeywell</td>
			<td></td>
			<td>Figure 7U</td>
		</tr>
		<tr>
			<td>Fly sleeper system with injector</td>
			<td>Inject + matic</td>
			<td></td>
			<td>Figure 6A-B</td>
		</tr>
		<tr>
			<td>Lamp power supply</td>
			<td>PTI</td>
			<td>LPS-220</td>
			<td>Figure 7W</td>
		</tr>
		<tr>
			<td>Light detector</td>
			<td>Home made</td>
			<td></td>
			<td>Phototransistor (Figure 7O)</td>
		</tr>
		<tr>
			<td>Light guide</td>
			<td></td>
			<td></td>
			<td>3 mm diameter, 1.3 m long (Figure 7L,M)</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td></td>
			<td></td>
			<td>High-pressure ozone-free 75 W Xenon lamp (operating on 50 W), possible replacement: Cairn research- OptoLED (Figure 7R)</td>
		</tr>
		<tr>
			<td>Low temperature melting wax</td>
			<td>Home made</td>
			<td></td>
			<td>Composed of mixture of beeswax (Tm&#8776;62 &#176;C) and paraffin at ~3:1 to reach a melting temperature of ~55&#8211;56 &#176;C (Figure 6J)</td>
		</tr>
		<tr>
			<td>Magnetic stand for flies</td>
			<td>Home made</td>
			<td></td>
			<td>Figure 6I and Figure 7Q</td>
		</tr>
		<tr>
			<td>Microelectrode preamplifier system with head-stage</td>
			<td>Almost perfect electronics</td>
			<td></td>
			<td>Impedance tester (Figure 7G)</td>
		</tr>
		<tr>
			<td>Micromanipulator (mechanical coarse)</td>
			<td>Tritech Research, Narishige</td>
			<td>M-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator (mechanical fine)</td>
			<td>Leitz Microsystems</td>
			<td>Leitz Mechanical Micromanipulator</td>
			<td>Figure 7F</td>
		</tr>
		<tr>
			<td>pCLAMP</td>
			<td>Molecular Device</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td></td>
			<td></td>
			<td>60 mm</td>
		</tr>
		<tr>
			<td>Pulse generator</td>
			<td>AMPI</td>
			<td>Master 8</td>
			<td>Figure 7A</td>
		</tr>
		<tr>
			<td>Redux cream for electrocardiography</td>
			<td>Parker Laboratories</td>
			<td>Redux Electrolyte Cr&#232;me</td>
			<td></td>
		</tr>
		<tr>
			<td>Shutter driver</td>
			<td>Uniblitz, Vincent Associates</td>
			<td>VCM-D1 Single Channel Uni-stable</td>
			<td>Figure 7V</td>
		</tr>
		<tr>
			<td>Shutter system</td>
			<td>Uniblitz, Vincent Associates</td>
			<td>LS2 2 mm Uni-stable Shutters</td>
			<td>Figure 7V</td>
		</tr>
		<tr>
			<td>Silver Wire</td>
			<td>Warner Instruments</td>
			<td></td>
			<td>0.25&#8211;1 mm diameter, needs to be chloridized</td>
		</tr>
		<tr>
			<td>Soldering iron composed of a platinum-iridium filament</td>
			<td></td>
			<td></td>
			<td>0.25 mm diameter (Figure 6F)</td>
		</tr>
		<tr>
			<td>Stereoscopic zoom Microscope</td>
			<td>Nikon</td>
			<td>SMZ-2B</td>
			<td>Figure 6D</td>
		</tr>
		<tr>
			<td>Stereoscopic zoom Microscope</td>
			<td>Wild</td>
			<td>Wild M5</td>
			<td>With 6, 12, 25 and 50 magnification settings (Figure 7E)</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Millex</td>
			<td></td>
			<td>22 &#181;m PVDF filter</td>
		</tr>
		<tr>
			<td>Vertical pipette puller</td>
			<td>Sutter/ Narishige</td>
			<td>Model P-97/PP-830</td>
			<td>Use either vertical or horizontal puller, as preferred (Figure 6L)</td>
		</tr>
		<tr>
			<td>Wax filament heater</td>
			<td>Home made</td>
			<td></td>
			<td>See figure S1 (Figure 6E-G)</td>
		</tr>
		<tr>
			<td>Xenon Flash Lamp system</td>
			<td>Dr. Rapp OptoElectronic</td>
			<td>JML-C2</td>
			<td>Figure 7X</td>
		</tr>
	</tbody>
</table>

electrophysiological methods for measuring photopigment levels in drosophila photoreceptors

果蝇G蛋白偶联的光色素沉着视紫红质（R）由蛋白质（视蛋白）和发色团组成。视紫红质的活化过程由光子吸收诱导发色团的异构化启动，促进视蛋白的构象变化并导致第二种暗稳定光色素沉着状态（偏视紫红质，M）。使用随机诱变来研究这种双稳定光色素需要简单而可靠的方法来筛选突变蝇。因此，已经设计了几种测量功能性光色素水平降低的方法。一种这样的方法利用了光子吸收后光色素内的电荷位移以及光感受器中表达的大量光色素分子。该电信号称为早期受体电位（或早期受体电流），通过各种电生理学方法（例如，视网膜电图和全细胞记录）测量，并且与功能性光色素水平成线性比例。该方法的优点是高信噪比，直接线性测量光色素水平，以及视紫红质或偏视红质激活下游的光转导机制的独立性。另一种称为延长去极化后电势（PDA）的电生理学方法利用果蝇光色素的双稳定性和苍蝇R和M色素状态的吸收光谱差异。PDA由强烈的蓝光诱导，将饱和量的视紫红质转化为偏视紫红质，导致在黑暗中长时间光响应终止失败，但它可以通过使用强烈的橙色光将偏视紫红质转化为视紫红质来终止。由于PDA是一种强大的信号，需要大量的光色素转化，因此即使是光色素生物发生中的微小缺陷也会导致容易检测到异常的PDA。事实上，有缺陷的PDA突变体导致了对光转导很重要的新型信号蛋白的鉴定。

图2说明了使用ERG技术的鲁棒性和易用性。它之所以坚固，是因为它通过一种简单的细胞外电压记录技术记录在几乎完整的飞行中，这需要简单的电生理设置。即使突变强烈降低或扭曲光响应，也通过以相对较大的振幅（在毫伏范围内）获得光响应的记录来表现鲁棒性。因此，即使是没有经验的实验者也可以使用非常简单的实验装置，并学习如何在几天内获得有意义的结果。...

Watch this Scientific Journal Video about Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors at JoVE.com

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

我们提出了一种电生理学表征双稳定光色素的方案：（i）利用光子吸收后光色素分子内的电荷位移及其在光感受器中的大量电荷，以及（ii）利用视紫红质和偏视紫红质光色素状态的吸收光谱差异。这些方案可用于筛选影响双稳定光色素系统的突变。

测量 果蝇 光感受器中光色素水平的电生理学方法

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

该方案特别适用于视觉缺陷果蝇突变体的遗传筛选。这是因为它们允许检测果蝇光转导中的微小异常，特别是在光色素水平和分层同伴相关功能方面。该技术在体内意义深远，易于执行且高度稳健，能够检测光转导蛋白活性的微小变化。在我看来，在保持生存能力的同时修复苍蝇可能是这种方法的关键部分。打开计算机并打开 Clampex 软件。打开放大器和 Master-8 脉冲发生器，然后打开氙气灯和快门控制器。打开拉拔器，在拉拔器中放置玻璃毛细管并拉动它。从拉拔器上取下玻璃毛细管。使用细长的尖端注射器用过滤的铃声溶液填充玻璃毛细管。将电极丝插入玻璃毛细管。确保毛细管内的溶液与银线接触。将电极支架插入两个电极显微操纵器中。打开烙铁的电源。将电流设置为大约 2.25 安培。该电流应将0.25毫米铂铱丝加热到约55至56摄氏度。将一滴熔化温度较低的蜡放在烙铁上。麻醉瓶内的苍蝇。将麻醉的苍蝇倒入卧铺容器中，选择一只苍蝇并用培养皿覆盖其余的苍蝇。用锋利的镊子小心地抓住苍蝇的翅膀，然后将其放在苍蝇支架上。在苍蝇支架上，将苍蝇侧卧，背部朝向手。用镊子将苍蝇从翅膀上抬起，然后用烙铁将其翅膀固定在苍蝇支架上。使用烙铁用蜡将苍蝇的背部连接到支架表面。将烙铁的尖端降低到腿的连接点，然后将蜡熔化以将所有腿覆盖在一起。在颈部区域的头部和颈部之间滴一小滴蜡。将苍蝇支架放在磁铁块上的黑暗法拉第笼中，并确保苍蝇距离光导末端约 5 毫米。使用显微操纵器将记录电极放在苍蝇的眼睛上方，将接地电极放在苍蝇的上背部。然后使用显微操纵器将接地电极插入苍蝇的背面。将记录电极插入苍蝇眼的外周，最好使用微型操纵器。关闭法拉第笼并关闭房间内的灯，以适应黑暗五分钟。输入从脉冲持续时间 500 米/秒、脉冲间隔 60 秒和脉冲数设置为 6 开始的参数。要测量PDA，请将Master-8脉冲发生器设置为TRAIN模式，然后使用橙色滤光片发出最大强度的五秒光脉冲。检查电压响应。用宽带蓝色滤光片替换橙色滤光片，并以最大强度发出三个 5 秒的光脉冲。检查电压响应。在黑暗中等待至少 60 秒。将蓝色过滤器替换为以前的橙色过滤器。然后以 60 秒的间隔给出两个 5 秒的光脉冲。检查电压响应。为了测量ERP M电位，请发出连续的蓝光脉冲，直到达到稳态电压响应。使用 20 纳米带通滤光片提供波长在 350 至 700 纳米之间的短暂强光闪光。然后测量M电位响应的M1相的峰值幅度，它反映了光平衡下该特定波长下间视紫红质的吸收。评估不同突变果蝇的PDA反应。野生型苍蝇在强烈的蓝光刺激后，在黑暗中表现出饱和的去极化，表现为长时间的角膜负ERG。以下蓝灯产生叠加在PDA上的小响应，缺少开和关瞬变。在具有异常光色素生物发生（如ninaE）的突变果蝇中，电压响应在强烈的蓝光刺激后恢复到基线，并且对额外蓝光的反应未受到抑制，并显示出正常的开关瞬变。苍蝇的野生型ERP是通过相同的协议获得的，但在PDA期间应用了强烈的绿色闪光。此绿色闪光触发 ERP 并抑制 PDA。M电位是通过蓝色照明后的绿色闪光获得的，而不是通过野生型苍蝇蓝色照明后的蓝色闪光获得的。该方案在野生型，光色素亚型突变体ninaE和具有正常光色素水平norpA的光转导缺陷突变体中重复。通过不同光谱和光平衡光谱的光度测量计算了苍蝇视紫红质和视紫红质的相对吸收光谱。对于苍蝇的生存来说，通过避免过热来注意蜡的粘度很重要。该程序适用于遗传和通过筛选有缺陷的视觉突变体。随机分离和识别视觉突变的方法可能有助于发现参与果蝇光转导的新蛋白质和机制。PDA协议能够隔离重要和新颖的视觉机制。否则，参与可能不会被发现，甚至没有预料到。

Research

JoVE Journal

Neuroscience

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

Methods to Assay Drosophila Behavior

Methods to Assay Drosophila Behavior

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

科研

神经科学

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

局部和全局的方法评估热痛觉<em&gt;果蝇</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

电生理记录从<em&gt;果蝇</em&gt; Labellar味感器

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受<em&gt;果蝇</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Measuring and Altering Mating Drive in Male Drosophila melanogaster

测量并改变配套驱动马累<em&gt;果蝇</em

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

测量 果蝇 光感受器中光色素水平的电生理学方法

测量果蝇光感受器中光色素水平的电生理学方法